Introduction:
Protein expression involves taking a vector and transforming a strain of bacteria with it [1]. The strain of E. coli BL21 (DE3) is a good strain because it is "fast and inexpensive to test." In order to isolate the protein, researches have developed a method called chromatography, which involves adding a Histidine tag in the protein which will bind to an ion and separate it from the other compounds [1].
In this experiment, a protein will be isolated by culturing transformed bacteria and purifying the protein through chromatography, and then identified with gel electrophoresis.
Materials & Methods:
Two culture plates of Escherichia coli (BL21 (DE3)) (New England BioLabs Inc., Ipswich, MA) were grown: one control plate and one experimental plate with the DNA plasmid (pGEM-gbr22). The experimental plate was transformed by heat shock.
After growth, the cultures were transferred to two flasks of 25 mL of LB broth and ampicillin. The next day, 500 μL of the culture was stored in a 1.7 mL Eppendorf tube as Sample 1. Both cultures were spun in a centrifuge and both pellets were weighed. PBS solution was added to the tubes and the tubes were vortexed. Lysozyme was added to each tube.
One of the tubes was thawed later and benzonase was added in and mixed. This lysate solution was centrifuged and 50 μL of the supernatant was stored as Sample 2. The rest of the supernatant was then put through a syringe filter into a 14 mL round bottom transformation tube.
0.5 mL of blue Ni-NTA was added to the supernatant. A 20 mL Bio-Rad chromatography Econo column was rinsed with Nanopure water. The resin and buffer were then transferred to the column and allowed to settle. After settling, the fluid was collected in a 10 mL round bottom tube labeled "Waste." 50 μL of this sample was taken and stored as Sample 3.
The Ni-NTA resin was washed with 20 mM imidazole in 1x PBS, collected, and labeled "Wash." 50 μL of this wash was stored as Sample 4.
5 mL Buffer containing 250 mM imidazole was added to the top of the column. Flow through was collected and labeled "Elution 1." Another 5 mL of elution buffer was added, collected, and labeled "Elution 2." A 50 μL sample of each elution was stored as Sample 5 and Sample 6, respectively. After, the column was stripped and stored with 30% ethanol.
A Nanodrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) measured the absorbance of Elution 1 at wavelengths of 280 nm and 574 nm in order to determine the concentration of protein.
Sample 1 was centrifuged first and then the pellet was retained and resuspended in 200 μL of Nanopure water. 40 μL of blue loading buffer was added to Sample 1, and 10 μL to the other five samples. The 6 samples that were taken were run in gel electrophoresis next to a lane of molecular weight standards. After the run, the gel was stained with Imperial protein stain and destained after. Finally, the gel was dried.
Results:
Figure 1: Escherichia coli on a control plate.
Figure 2: E. coli transformed with pGEM-gbr22.
Figure 3: Plate of bacteria from human cough.
Figure 4: Flask of transformed E. coli. Sample 1 was taken from this.
Figure 5: Resuspended pellet in PBS solution after centrifugation. The pellet itself weighed 0.59 g.
Figure 6: Elutions 1 (right) and 2 (left).
Figure 7: Absorbance of Elution 1 at 280 nm. Trial 1.
Figure 8: Absorbance of Elution 1 at 280 nm. Trial 2.
Figure 9: Absorbance of Elution 1 at 574 nm. Trial 1.
Figure 10: Absorbance of Elution 1 at 574 nm. Trial 2.
Yields
280 nm: 2.34 mg of protein
574 nm: 1.19 mg of protein
Figure 11: Protein Gel. 1) Molecular weight standards. 2) Sample 1: Entire cells. 3) Sample 2: Supernatant after centrifugation. 4) Sample 3: Waste flow through. 5) Sample 4: Washed protein. 6) Sample 5: Elution 1. 7) Sample 6: Elution 2. Lanes 8, 9, and 10 are repeats of lanes 5, 6, and 7.
In sample 5, there is 1 solid band. This is the protein that is being identified.
Figure 12: Molecular weight standards used in lane 1.
Based on these standards, the molecular weight of the protein is about 29 g.
Discussion: The results were fairly precise. Based on the gel, the filter and elution process proved accurate with the darker band going straight across the gel. Unfortunately, elution 1 in lane 6 had many more structures in it than just the protein. This might result in not enough Ni-NTA placed in the column.
Lysozyme was used to break down the cell membrane and release all the contents of the cell. Benzonase was used to digest DNA/RNA in the mixture, which made the solution more viscous.
The wash buffer and elution buffer differ in the concentration of imidazole. Imidazole plays a role in the HIS tag system. The HIS tags in the protein have a strong affinity for the Nickel ion in Ni-NTA. However, by increasing the concentration of imidazole, the imidazole binds to nickel instead of the protein, allowing the protein to flow through.
Conclusions: In this lab, bacteria were transformed and the purple protein that was produced was isolated and identified. Processes such as chromatography, spectrophotometry, and gel electrophoresis were performed. Ultimately, we found the concentration of the protein in the extracted liquid as well as its molecular weight based on gel electrophoresis.
This lab is a small example of how proteins can be identified in bacteria in order to see if they are expressed by the cell. Researchers can use this method to identify different proteins in bacteria. Imidazole can be used to identify other proteins with HIS tags. Proteins can be singled out based on their characteristics.
Introduction:
Protein expression involves taking a vector and transforming a strain of bacteria with it [1]. The strain of E. coli BL21 (DE3) is a good strain because it is "fast and inexpensive to test." In order to isolate the protein, researches have developed a method called chromatography, which involves adding a Histidine tag in the protein which will bind to an ion and separate it from the other compounds [1].
In this experiment, a protein will be isolated by culturing transformed bacteria and purifying the protein through chromatography, and then identified with gel electrophoresis.
Materials & Methods:
Two culture plates of Escherichia coli (BL21 (DE3)) (New England BioLabs Inc., Ipswich, MA) were grown: one control plate and one experimental plate with the DNA plasmid (pGEM-gbr22). The experimental plate was transformed by heat shock.
After growth, the cultures were transferred to two flasks of 25 mL of LB broth and ampicillin. The next day, 500 μL of the culture was stored in a 1.7 mL Eppendorf tube as Sample 1. Both cultures were spun in a centrifuge and both pellets were weighed. PBS solution was added to the tubes and the tubes were vortexed. Lysozyme was added to each tube.
One of the tubes was thawed later and benzonase was added in and mixed. This lysate solution was centrifuged and 50 μL of the supernatant was stored as Sample 2. The rest of the supernatant was then put through a syringe filter into a 14 mL round bottom transformation tube.
0.5 mL of blue Ni-NTA was added to the supernatant. A 20 mL Bio-Rad chromatography Econo column was rinsed with Nanopure water. The resin and buffer were then transferred to the column and allowed to settle. After settling, the fluid was collected in a 10 mL round bottom tube labeled "Waste." 50 μL of this sample was taken and stored as Sample 3.
The Ni-NTA resin was washed with 20 mM imidazole in 1x PBS, collected, and labeled "Wash." 50 μL of this wash was stored as Sample 4.
5 mL Buffer containing 250 mM imidazole was added to the top of the column. Flow through was collected and labeled "Elution 1." Another 5 mL of elution buffer was added, collected, and labeled "Elution 2." A 50 μL sample of each elution was stored as Sample 5 and Sample 6, respectively. After, the column was stripped and stored with 30% ethanol.
A Nanodrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) measured the absorbance of Elution 1 at wavelengths of 280 nm and 574 nm in order to determine the concentration of protein.
Sample 1 was centrifuged first and then the pellet was retained and resuspended in 200 μL of Nanopure water. 40 μL of blue loading buffer was added to Sample 1, and 10 μL to the other five samples. The 6 samples that were taken were run in gel electrophoresis next to a lane of molecular weight standards. After the run, the gel was stained with Imperial protein stain and destained after. Finally, the gel was dried.
Results:
Yields
280 nm: 2.34 mg of protein
574 nm: 1.19 mg of protein
In sample 5, there is 1 solid band. This is the protein that is being identified.
Based on these standards, the molecular weight of the protein is about 29 g.
Discussion:
The results were fairly precise. Based on the gel, the filter and elution process proved accurate with the darker band going straight across the gel. Unfortunately, elution 1 in lane 6 had many more structures in it than just the protein. This might result in not enough Ni-NTA placed in the column.
Lysozyme was used to break down the cell membrane and release all the contents of the cell. Benzonase was used to digest DNA/RNA in the mixture, which made the solution more viscous.
The wash buffer and elution buffer differ in the concentration of imidazole. Imidazole plays a role in the HIS tag system. The HIS tags in the protein have a strong affinity for the Nickel ion in Ni-NTA. However, by increasing the concentration of imidazole, the imidazole binds to nickel instead of the protein, allowing the protein to flow through.
Conclusions:
In this lab, bacteria were transformed and the purple protein that was produced was isolated and identified. Processes such as chromatography, spectrophotometry, and gel electrophoresis were performed. Ultimately, we found the concentration of the protein in the extracted liquid as well as its molecular weight based on gel electrophoresis.
This lab is a small example of how proteins can be identified in bacteria in order to see if they are expressed by the cell. Researchers can use this method to identify different proteins in bacteria. Imidazole can be used to identify other proteins with HIS tags. Proteins can be singled out based on their characteristics.
References:
1. http://www.embl.de/pepcore/pepcore_services/index.html
2. Nat Methods. 2008 Feb;5(2): 135-46. Protein production and purification.